4.1 Protein Crystallization Approaches
Protein expression and purification is an expensive process, and it is sometimes difficult to express proteins in large quantities. A large amount of expensive protein is required to establish conditions for crystallization. Consequently, there is a need to reduce the amount of protein required for crystallization screening and to do it more efficiently and at lower cost.
4.1.1 Protein Crystal Synthesis
Proteins play a key role in all biological processes. The specific biological function of a protein is determined by the three-dimensional (3D) arrangement of the constituent amino acids. Understanding a protein's 3D structure plays an important role in protein engineering, bioseparations, rational drug design, controlled drug delivery, and the design of novel enzyme substrates, activators, and inhibitors. Protein crystallization is a multi-parametric process that involves the steps of nucleation and growth, during which molecules are brought into a thermodynamically unstable and a supersaturated state.
4.1.2 Miniaturization and Automation of Protein Crystallization Setup
Many proteins of interest are unfortunately available only in limited supply. Efforts are ongoing to reduce the consumption of proteins by miniaturizing the crystallization setup. Despite efforts to reduce the protein volumes, these processes still consume significant amount of protein and are still labor-intensive.
Existing semi-automatic systems do not encompass ideal high-throughput configurations. They require user intervention for multiple tray processing and have other material processing issues. As most of the work performed with these systems is not on a large scale, automation of storage and handling of plates was not addressed in these systems. Even though these industrial systems are capable of setting up thousands of crystallization screens a day, they are prohibitively expensive for academic research labs. There remains a need in the art for a system that provides the high-throughput automation functionality of an industrial system at an affordable cost for small laboratories or individual investigators.
4.1.3 Lab-on-a-Chip Technologies
Microfluidic systems can be broadly categorized into continuous-flow and discrete-flow based systems. As the name suggests, continuous-flow systems rely on continuous flow of liquids in channels whereas discrete-flow systems utilize droplets of liquid within channels or in an architecture without channels. A common limitation that continuous flow systems face is that liquid transport is physically confined to fixed channels. The transport mechanisms used are usually pressure-driven by external pumps or electrokinetically-driven by high-voltages. These approaches involve complex channeling and require large supporting instruments in the form of external valves or power supplies. These restrictions make it difficult to achieve high degrees of functional integration and control in conventional continuous-flow systems.
4.2 Multi-Well Plates
Microfluidic technologies are attracting attention in pharmaceutical research, as miniaturization of assay volume and improvement of automation, throughput and precision become more critical in drug discovery research. Examples of recent microfluidic technologies and products include the Topaz™ system for protein crystallization from Fluidigm Corporation (San Francisco, Calif.), the LabChip® system from Caliper Life Sciences (Hopkinton, Mass.), and the LabCD™ system from Tecan Systems Inc. (San Jose, Calif.), both for ADME. These systems perform certain assays using small volumes of liquid. However, none of them even remotely approaches the flexibility of conventional robotic systems. This inadequacy results from inherent technical limitations associated with the way in which fluid handling is implemented in these devices.
Most existing technologies are based on a continuous-flow approach. Liquid is pumped (generally unidirectionally) through a network of microchannels using external pumps, valves, high-voltage supplies or centrifugal force. The primary disadvantage of all of these continuous-flow microfluidic devices is their architectural and operational rigidity. Most are optimized for a particular assay, providing little or no flexibility to make changes in reaction protocols. The required continuity of fluid in these devices also makes independent operation of different areas of the chip an inherently difficult proposition. Consequently, these technologies are non-modular and difficult to scale.
There is a need in the art for a microfluidic platform that avoids the use of a continuous-flow approach. There is a need for a system that affords flexibility and programmability that is comparable to robotic systems. Further, there is a need for a system that is capable of working with droplets as small as a few nanoliters in volume and avoids the requirements for a network of microchannels, external pumps, valves, high-voltage supplies and/or centrifugal force. Further, there is a need for a system that is scalable, permitting hundreds or even hundreds of thousands of droplets of liquid to be processed in parallel. Finally, there is a need for a system that is both compact and inexpensive to manufacture.
4.3 Protein Stamping Platforms
Mass spectroscopy (MS) is increasingly becoming the method of choice for protein analysis in biological samples. Among the various MS methods, MALDI-TOF (Matrix Assisted Laser Desorption-Ionization Time of Flight) is the most commonly used due to its simplicity, high sensitivity and resolution. A typical MALDI-MS protocol for protein identification involves sample preparation, stamping onto a MALDI target and analysis on a MALDI-TOF mass spectrometer. Sample preparation steps (such as digestion and concentration) are usually done in the well-plate format and last for several hours at the least. The stamping is accomplished using complex robotic systems which are huge, expensive and immobile. Required sample volumes are also very high, which is a concern for proteins available in very small quantities. Existing microfluidic devices are based on continuous flow in fixed microchannels, offering very little flexibility in terms of scalability and reconfigurability. As such, a need exists for a droplet microactuation stamping platform designed to solve the deficiencies found in the prior art.